Fig. 5. Stability of 5heC against hydrolytic enzymes. (A) Schematic showing coupled assay involving deamination and base excision of FAM-labeled DNA. (B) TDG can excise both ; 5hmU and ; 5heU duplexed DNAs carrying a U:G mismatch as confirmed by in-gel fluorescence. (C) Activity of wild type AID and its Y116A mutant towards DNA strands carrying either ; 5hmC (R = H) or ; 5heC (R = CH3) modifications. Deaminated DNAs were duplexed to create U:G mismatch prior to TDG-mediated base excision and strand cleavage. (D) Crystal structures of human AID (PDB: ; 5w0u). Y116 resides closely to substrate cytidine in the active site. (E) Sequence alignment showing that Y116 is conserved in human AID and APOBEC family of deaminases. (F) The tyrosine is likely acting as a gatekeeper residue that can be mutated to smaller amino acid to accommodate modified cytidine. S: substrate DNA; P: product DNA; dCMP: deoxycytidine monophosphate.
